Process of producing butyl alcohol and acetone by fermentation



Patented Feb. 14, 1939 UNITED STATES PROCESS OF PRODUCING BUTYL ALCOHOL AND ACETONE BY FERMENTATION Alfred Frey, Freising, and Hans Gliick, Munich,

Germany No Drawing. Application April 21, 1937, Serial No. 138,186. In Germany March 16, 1936.

. scam The bacterial cultures used in the process of producing butanol and acetone generally pass through a definite cycle of development, during which they convert carbohydrates into butyric 5 acid and acetic acid, and then reduce these to.

butanol and acetone, the reduction being in part immediate.

When these cultures, which include Clostri- 'dium acetobu-tylicum and various morphologically 10 similar butanol formers, are further developed it has been noted, e. g., by Bernauer (Biochemische Zeitschrift, Nr. 280, page 383; 1935) that there are signs of degeneration, including reeiced resistance to acid and reduced de-oxidizing power. .5 In the course of normal fermentation, with formation of Clostridia and spores, there is a re duction of acid with abundant production of butanol, whereas with a degenerated culture there is a continuous increase of acid, without appearance of Clostridia and spores, and the breaking down of the sugar ultimately stops and cannot be re-started by neutralizing the acid with lye or lime, though, the contrary was held by F. B. Fred, W. H. Petersen and M. Mulvania (Journ. Bact. No. 11, page 323).

The following tablebriefiy indicates the course of two butanol fermentations, withcultures at the same stage of development in the same medium (potato mash 6 B1lg.):

Normal fermentation lg ffi m' Acid ccni. I Acid ccm. 5 38$? n/l NaOH/ZO cc. $3, 53, n/l NaOH/20 a.

mash filtrate mash filtrate The content of solvents such as butanol, ace-.

L produce'a pH value of from 3.65 to 3.75 there (on. 19 4s) was complete stoppage of butanol-acetone fermentation.

Degeneration is to be observed when laboratory cultures are bred under aerobic or anaerobic conditions, with or without change of medium. 5 It occurs also in the case of cultures which appear to be normal but are weakened by breeding and are then transferred to a larger quantity of mash.

There have been several proposals for preventing this occurrence and the consequent loss. It has for instance been proposed to pre-heat the mash so as to destroy vegetable organisms therein, and it was stated that where this is done and a particular bacterium is used (Bacillus butylicus Boinot-Firmin) there was no failure of fermenlit tation, even after prolonged breeding.

It is a common practice in bacteriology to heat'mixtures of bacteria, so as to separate heat resistant spores from less resistant spores. This has been done in order to procure and develop more emcient breeds. Definite precautions are, however, necessary! Heating butyl bacteria to 100 C. weakens the culture,

A proposal not hithereto published is that of heating the cultures in an alkaline culture me-' 25 dium, for the purpose of preventing degeneration.

We have found that forpreventing defective fermentation with a high degree of reliability it is necessary to carry out the proposed treatments in a particular sequence and a particular manner. Neither heating alone, nor transfer to an alkaline medium, is in itself sufiicient to ensure a thoroughly sound culture. We have found, in particular, that it is not correct to efiect the elimination of vegetable forms, by heating, in the alkaline medium. It should be done in an interposed normal stage. In the alkaline medium the spores are too sensitive to heat, so that in addition to killing the vegetable forms the heat weakens the spores. As regards adding lime, this must'be so carried out that there is an alkaline reaction during the whole period of development. The process has the advantage of simplicity, enabling cultures to be bred in the laboratory, which can subsequently be used for large scale work and transferredfrom mash to mash with retention of good fermenting properties. The heating is neither so long as to weaken the cultures, nor does it take place in an alkaline medium conducive to weakening.

Example I From 5 to 10 com. of a culture of Clostridium acetobutylicum aresealed in a tube with 15 com. of a 6 Balling potato mash, the sealing being effected with paraffin wax softening at from 60 to 65 C. The tube is heated for two minutes in boiling water. We have found two minutes to be the optimum heating period. Then the tube is quickly cooled and well shaken, and is kept for two days at a temperature of 37.5 C. After '48 hours, following this treatment, the culture is found still to consist mainly of short rods, A transfer is then made to a second tube, where 0.25 gr. CaCOs are added, keeping the pH value above 7.1 throughout a period of 48 hours, when the whole culture will consist of long and short rods. Then another transfer is made to a tube without addition of lime, but'with heating, and

after .!8 hours the appearance of Clostridia is 'observed. This alternation-frofn normal culture to lime-treated culture may be repeated three or four times with increased appearance of 0105- tridia and spores after 48 hours. We have found thatwith a culture thus developed the twentythird generation still produced normal fermenvtation. carried out as follows:

5 litres of 6 Bailing mash the culture of Clostridium dcetobutulicum in the twenty-fourth stage of generation, and were kept- 72 hours at 87.5 C. The following table shows the result:

. Before o fermentafermentation tion em mum (degrees Belling)"; o 1. 25 AgdltyoimsshflnmnflNaOH/mcan.) 2 lg n a Bolvonts in fermented mash.-.-

........ ............volume, per cen 2. 05'

, sample n The same quantity and kind of mash was treated with a culture developed with only seven transfers from normal tubes to tubes with alkaline medium, and the mesh was kept for '12 'hoursat37.5 C:

am Ame fermentfermentation. tion 'ooncsnnationvdogressll'allin o u Aeidltyofmssls .1) 1.4 an as The not only adapted to prevent degeneration of the cultures, but also to restore degenerated cultures to normal efficiency for fermentation. Y

seam zeur A receptacle of fermentation with a content of 20 litres of. potato mash is insrafted with soov ccm. of a first ferment, made according the Examlfles I and'IL After finish of the main fermentation phase, Vsof this mash (4 litres) is given in 10 litres of a fresh mash. This 1378.-

. peatedH-times in'sucoession; The remaining nubes'srenbrmal fermentated through were treated with v in this manner.

2. The herein described 'process of producing 2nd phase 2.55 .vol. butanol-acetone 3rd phase 2.01 vol. butanol-acetone llfedium of the 5 phases -2.60 vol, This form of the transfer at the moment of the beginning formation of Clostridia has opposite to others described continuous processes of fermentation the advantage that the bacteria can acomplish their natural cycle of acretion and a certain reduction of the nascent butyric acid into butanol. What we claim is: T

1. The'herein described process of producing butyl alcohol and acetone by fermentation, which process comprises the steps of heating cultures of butanol forming bacteria in a normal culture medium to the'boiling temperature of water, transferring said cultures from said normal medium to an alkaline medium with a pH value kept above 7.1 by addition of alkali, the

bacteria being bred in said alkaline medium at normal aeration temperature'of about 37.5 C., repeating the heat treatment in the normal medium and the breeding step in the alkaline medium, and inoculating a mash with bacteria bred butyl alcohol and acetone by fermentation, which process comprises the steps of subjecting cultures of butanol forming bacteria, in a normal culture medium for two minutes to a heat treat-.

ment in boiling water, transferring said cultures from said normal medium to an alkaline medium with a pH value kept above 7.1 by addition of alkali, the bacteria being bred in said alkalinemedium at'no'rmal aeration temperature I of about 375 C.,' repeating the heat treatment in the normal medium and the breeding step with bacteria bred in this manner.

3. The herein described process of producing butyl alcohol and acetone by fermentation, which process comprises the steps of heating cultures of butanol forming bacteria in a normal culture mediumto the boiling temperature of water,

-' inoculating a quantity of mash with bacteria bred in this manner, fermenting said mash until the vegetative generation of bacteria ceases,

' transferring a fraction of the fermented'mash to -a quantity of fresh, mash supplementing said fraction to the initial quantity, fermenting said fresh mash and repeating severaltimes the transfer of a fraction of fermented mash to a quantity of fresh mash and the fermentation of the latter.

muss anflcx.

in the alkaline medium, and inoculating a mash v 

